Hey @colinbrislawn . On this same topic, I wonder what you suggest when sequence quality drops off markedly before the R1 and R2 reads will overlap. For example, check out this demux stats ( online , and the raw file (312.8 KB)). These were 300 bp PE reads from a MiSeq, but quality really drops off af…

Hello Alex, [image] alexkrohn: Is there ever a time where you would suggest analyzing these data as a SE run? E.g. only analyze the 150bp of the R1 reads? I think your data set shows the exact time in which you would have to only use one of the reads because joining is impossible due to low …

The amplicon is 357 bp long, so with 300 bp PE sequencing, we should expect nearly total overlap without trimming. However, trimming down to 150-160bp with just the R1 would miss almost half the gene. If I do need to trim each read to 150bp, it seems like it would make sense to analyze both the R1 a…

[image] Seleting trunc length to allow Dada2 merge If I do need to trim each read to 150bp, it seems like it would make sense to analyze both the R1 and the R2 separately (e.g. as two SE runs). Does that make sense to you? Yes, that makes sense to me. With those quality scores, I think that's …

Thanks for your input! I'll give this a shot. Do you think it's worth combining the R1 and R2 reads (without merging them into one read) into one rep-seqs.qza file to train a taxonomy classifier/identify taxonomic units? Or just identify taxonomic units with the R1 and R2 reads separately? [imag…

[image] alexkrohn: Do you think it's worth combining the R1 and R2 reads (without merging them into one read) into one rep-seqs.qza file to train a taxonomy classifier/identify taxonomic units? This is possible in theory, but in practice, there are many unexpected implications of working with…

Hey @colinbrislawn . After some digging, it seems that these quality issues are just due to the nature of the eDNA that the data were derived from. I was wondering if I might change the error acceptability threshold in the dada2 denoise step to possibly include more data. I see that --p-max-ee is set…

I think max-ee (maximum expected errors per read) of 2 is pretty high already, as DADA2 could resolve ASVs down to a single difference/error. More info on expected errors and why just truncating at a low q-score is a bad idea . It might be worth trying a bunch of truncation settings to see if you c…

Got it! I'll try the truncation settings. Thanks again!

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I wonder what you suggest when sequence quality drops off markedly before the R1 and R2 reads will overlap

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